Pseudomonas aeruginosa is a Gram-negative bacterium responsible for a wide range of infections in humans. It is the leading cause of ventilator-associated pneumonia and also an important pathogen in cystic fibrosis patients. The type III secretion system of P. aeruginosa, a major virulence determinant in acute infections, secretes four effector proteins into the cytoplasm of host cells: ExoU, ExoS, ExoT, and ExoY. Of these, ExoU is the most potent and associated with worse outcomes. Understanding how ExoU enables P. aeruginosa to cause severe pneumonia will aid development of therapeutics for prevention, management, and treatment of these infections. Preliminary data show that phagocytic cells, particularly neutrophils and macrophages, are frequently injected with ExoU during early pneumonia. In the absence of ExoU, phagocytes successfully eradicate relatively low inocula of P. aeruginosa bacteria from the lungs over the first 24 hours of infection; however, secretion of ExoU is accompanied by bacterial persistence in the lungs and eventually death of the host. This suggests that phagocytes are a functionally important target of ExoU during early pneumonia and that impairment of phagocyte function by ExoU leads to disease progression. This project will define how ExoU inhibits phagocyte function and determine whether ExoU intoxication of phagocytes is an important contributor to disease severity in a mouse model of acute pneumonia. In vitro and in vivo assays will be used to determine which stage of normal bacterial clearance (phagocytosis, bacterial killing, or phagocyte survival) impaired by ExoU. Experiments testing the impact of phagocyte intoxication on disease severity will utilize the following property of ExoU: two overlapping portions of the ExoU protein are individually non-toxic but together recapitulate cytotoxicity. Transgenic mouse technology and the Cre/LoxP system will be used to generate mice expressing the first portion of ExoU in either all cell types, all cells types except phagocytes, or in phagocytes only. Infection with a P. aeruginosa strain secreting only the second portion of ExoU will be performed to determine the role of phagocyte intoxication in disease progression. Completion of these experiments will determine the consequences of ExoU injection into phagocytes during P. aeruginosa infection and how this contributes to the pathogenesis of severe pneumonia.